Single-molecule array-based approaches to genome sequencing have the potential to deliver a dramatic reduction in sequencing costs over bulk methods by reducing reagent costs, increasing read lengths, and substantially decreasing the input DNA requirements. The latter advantage is critical when information retrieved from the sequencing technique sheds light on epigenetic modifications and damaged DNA bases, which cannot be amplified. Pacific Biosciences has developed and commercialized a single-molecule real- time (SMRT) sequencing instrument that utilizes as substrates an array of zero-mode waveguides (ZMWs), or nanoscale holes through an opaque metallic film. The ZMW array enables multiplexed DNA sequencing by employing single DNA polymerases for sequencing by synthesis, and detection is achieved using 4-color fluorescence microscopy and dye-labeled nucleotides. However, two major challenges currently limit the cost reduction and the capabilities of this technology. The first is the low yield of functional ZMWs that contain exactly one DNA polymerase molecule, as required for sequencing. The second is the reliance on diffusion of polymerase/template complexes into ZMWs, which results in sub-optimal loading into the array and increases the DNA input requirement. Despite the demonstrated ability of SMRT sequencing to identify DNA base modifications, high DNA inputs required preclude the gathering of epigenetic sequence data from native DNA, important for understanding the role of DNA modifications in aging and various diseases. In this proposal we present an innovative approach for reducing the cost of SMRT DNA sequencing, as well as enabling genomic and epigenomic sequencing from picogram levels of DNA without prior amplification. Our approach is to replace the glass bottom of each ZMW with a thin membrane that contains a single nanopore. The nanopore will serve two major purposes: 1) Precise positioning of individual DNA polymerases in ZMW arrays with greatly improved yields and controlled stoichiometry; 2) Orders of magnitude enhancement of the loading rates of input DNA that is to be sequenced into the ZMWs. To achieve our goals we have assembled a multidisciplinary team of investigators with expertise in cutting edge fabrication techniques, nanoscale science, and molecular biology. We divide our goals into aims that include the fabrication of the ZMW-pore devices, anchoring of individual DNA polymerases in ZMW- pore arrays, and the reduction of input DNA requirements by active DNA loading into the ZMW-pore devices. Finally, we will demonstrate using our ZMW-pore arrays sequencing of control DNA libraries, as well as direct epigenomic sequencing from picogram levels of human brain mitochondrial DNA. Our approach, if successful, will dramatically reduce the cost of DNA sequencing and pave way to massive high-throughput genomic and epigenomic sequence data.